Received: 7 June 2016 | Revised: 15 November 2016 | Accepted: 18 November 2016

DOI 10.1002/ajpa.23147

AJPA YEARBOOK ARTICLE

Beyond food: The multiple pathways for inclusion

of materials into ancient dental calculus

Anita Radini1 | Efthymia Nikita2 | Stephen Buckley1 |

Les Copeland3 | Karen Hardy4,5

1
BioArCh University of York, York, UK
2
Abstract
Science and Technology in Archaeology
Research Centre, The Cyprus Institute, Dental calculus (mineralized dental plaque) was first recognised as a potentially useful archaeologi-
Nicosia, Cyprus cal deposit in the 1970s, though interest in human dental calculus as a resource material has
3
School of Life and Environmental Sciences, increased sharply in the past few years. The majority of recent research has focused on the
University of Sydney, NSW 2006, Australia retrieval of plant microfossils embedded in its matrix and interpretation of these finds as largely
4
ICREA, Pg. Lluís Companys 23. 08010 the result of deliberate consumption of plant-derived food. However, while most of the material
Barcelona
described in published works does represent food, dental calculus is in fact a “depositional envi-
5

ria, UAB, Campus
Departament de Prehisto
ronment” as material can enter the mouth from a range of sources. In this respect, it therefore
UAB. 08193 Cerdanyola del Vallès
represents an archaeological deposit that can also contain extensive non-dietary debris. This can
Correspondence
Anita Radini BioArCh University of York, comprise a wide variety of cultural and environmental material which reaches the mouth and can
York, UK. become embedded in dental calculus through alternative pathways. Here, we explore the human
Email: anita.radini@york.ac.uk behaviors and activities besides eating that can generate a flux of particles into the human mouth,
Karen Hardy
the broad range of additional cultural and environmental information that can be obtained through
ICREA, Pg. Lluís Companys 23. 08010
Barcelona; Departament de Prehisto
ria, the analysis and contextualisation of this material, and the implications of the additional pathways
UAB, Campus UAB. 08193 Cerdanyola del by which material can become embedded in dental calculus.
Vallès.
Email: khardy@icrea.cat
KEYWORDS
dental calculus, diet, environment, microfossils

1 | INTRODUCTION
Dental calculus results from the calcification of plaque and can be
retrieved from skeletal material dating to most archaeological periods
(Figure 1). Indicative of its preservation potential is the fact that it has
been found on the teeth of a Miocene Sivapithecus dating to between
12 and 8 million years ago (Hershkovitz et al., 1997) as well as on late
Pliocene hominins (Blumenschine et al., 2003). It has become increas-
ingly clear that dental calculus is a store of a wide range of in situ bio-
graphical information, as it acts as a trap for microscopic fragments of
debris. These debris may include human cells, mineralized bacterial
structures and plant microfossils as well as chemical and biomolecular
compounds that have passed through the mouth during life (Buckley,
Usai, Jakob, Radini, & Hardy, 2014; Blondiaux & Charlier, 2008;
Charlier et al., 2010; Dobney, 1994; Dobney & Brothwell, 1987; Hardy
et al., 2012, 2015; Vandermeersch et al., 1994; Warinner, Speller,
Collins, & Lewis, 2015). This has led, unsurprisingly, to a growing inter-
est in dental calculus as a source of direct evidence for ancient bio-
graphical and dietary information.
As dental calculus forms in the human mouth, the assumption so
far has primarily been that much of this material represents food con-
sumed, and therefore offers direct information on items that were
intentionally eaten (e.g., Radini, Nikita, & Shillito, 2016). However, it is
now becoming increasingly clear that the material found embedded in
dental calculus can come from a range of different sources. The calcu-
lus matrix may be enriched with plant and other remains derived from
accidental inhalation or ingestion of non-dietary debris, oral hygiene
activities and use of the mouth as a third hand (e.g., Blatt, Redmond,
Cassman, & Sciulli, 2011; Blondiaux & Charlier, 2008; Buckley et al.,
2014; Charlier et al., 2010; Hardy, 2016; Hardy et al., 2016; Radini,
Buckley, et al., 2016). The need to be aware of the complex interaction
between people and plant resources when reconstructing plant use in
past human activity has been discussed previously (Beck & Torrence,
2006); however, though non-dietary debris in particular can be

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C 2017 American Association of Physical Anthropologists | 71
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72 | RADINI ET AL.

FIGURE 2 Micro CT scan of dental fragment of calculus (from

Hardy et al., 2013)

corynebacteria). When food is chewed, plaque is formed by the

FIGURE 1 Human dental calculus on a male individual from the
Medieval Parish of St. Michael’s, Leicester 1250–1400 AD
challenging to identify, there is a need to consider all possible pathways
for the inclusion of materials into dental calculus, in addition to deliber-
ate ingestion.
In the light of this, we examine the use of microfossils extracted
from dental calculus in ancient dietary reconstruction, and we
explore the many non-dietary pathways whereby micro-debris can
become embedded. We highlight the need to contextualise all results
adsorption of proteins and bacteria, predominantly the facultative
anaerobe Streptococcus mutans (Marcotte & Lavoie, 1998). These
metabolise salivary sugars to form a film that adheres to the surface of
the tooth. The bacteria are rapidly replaced by calcium phosphate salts
and if the plaque is not cleaned off, microorganisms nearest the tooth
surface ferment sugars in the saliva and produce acids that
demineralise the tooth, ultimately producing caries (Hillson, 2005). The
plaque hardens rapidly, beginning with the cell walls of the bacteria
(Hillson, 2005) and can be fully calcified within 2 weeks (Lieverse,
1999). The rough surface of dental calculus serves to attract other
bacteria which adhere to those already attached, while the gaps
between the cells are filled by other components of the saliva to
produce a layered structure (Figure 2) (Dobney & Brothwell, 1987;

within their natural and cultural environments and we conclude by Hardy, van de Locht, Wilson, & Tugay, 2013). The build-up of dental

suggesting that the real value of dental calculus, as a store for a wide calculus can be greatest near the salivary ducts; this results in deposits

range of biographical and environmental data, has yet to be fully that are more prominent on the lingual surfaces of incisors and canines

exploited. and the buccal surfaces of maxillary molars (Hillson, 2005). However,
calculus is not restricted to these locations and in extreme cases it can
almost cover all teeth.
2 | FORMATION PROCESS, STRUCTURE
When food is introduced into the mouth, the mechanical process
AND COMPOSITION OF DENTAL CALCULUS
of chewing can lead to material becoming entrapped in the gingival
crevice of the teeth (Figure 3). The bacteria in the gingival crevice are
2.1 | Formation process
predominantly proteolytic (utilising protein as substrate rather than
Dental calculus forms on teeth through a complex interaction
between saliva and bacteria on the dental surface. Saliva is made up
of water (99.5%), electrolytes, mucus, antibacterial compounds,
enzymes and bacterial cells (Roberts, 1979). Saliva lubricates the
mouth and moistens food to create a bolus, which is then swallowed.
It contains the enzyme a-amylase, which commences the breakdown
of starch into simple sugars and also immunoglobulins, which control
the microorganisms in the mouth and can restrict the build-up of pla-
gio, Machado, de Lima, & Azevedo, 2008). Saliva
que (de Almeida, Gre
is primarily formed by three pairs of glands, the parotid glands, the
submandibular glands and the sublingual glands, and empties into the
mouth through their respective ducts. The parotid gland ducts are
located inside the cheeks near the upper molars, while the subman-
dibular and sublingual gland ducts are located under the tongue
(Edgar, O’Mullane, & Dawes, 1996).
The mouth contains many species of Gram-positive and Gram-
negative bacteria (Hillson, 2005), including spherical cocci (e.g., strepto-
cocci and staphylococci), and rod-shaped bacilli (e.g., lactobacilli and FIGURE 3 Location of subgingival crevice on teeth

RADINI ET AL.
sugars, contrary to those in the mouth, which are saccharolytic); this
favors the preservation of starch granules once they have reached the
gingival crevice (Marcotte & Lavoie, 1998). The resulting metabolic by-
products of proteolytic metabolism, such as ammonia, result in local-
ized raised pH, which in turn favors plaque mineralization by stimulat-
ing precipitation of calcium phosphate. The antimicrobial constituents
of the saliva (e.g., immunoglobulins and degradative enzymes) break
down the microorganisms that have survived in the mouth (Marcotte &
Lavoie, 1998). However, some material escapes breakdown and can
become covered with plaque within hours. It is then protected from
the effects of the a-amylase and becomes embedded in the calculus
matrix (Marcotte & Lavoie, 1998; Scannapieco, Torres, & Levine,
1993).
Dental calculus has a multicausal etiology. Until recently, it was
widely assumed that calculus deposits indicated diets rich in protein
since such diets increase oral alkalinity, which in turn facilitates calculus
formation (Hillson, 1979; Lillie & Richards, 2000; Meiklejohn & Zvelebil,
1991; but see Lieverse, 1999 for a review of calculus formation proc-
esses). It is now clear that the dietary information provided by the
extent of calculus deposits is not as straightforward as originally
assumed. Specifically, high calculus deposition combined with low inci-
dence of caries has been considered to suggest a high protein intake
(Keenleyside, 2008; Lillie, 1996), whereas occurrence of both high cal-
culus and caries is understood to predominate in diets high in carbohy-
drates (Humphrey et al., 2014; White, 1994). In any case, non-dietary
factors such as the rate of salivary flow, mineral and silicon content
consumed in food and water, phosphate and calcium levels in the blood
and genetic factors also influence the occurrence of calculus deposits,
possibly more so than diet (Lieverse, Link, Bazaliiskiy, Goriunova, &
Weber, 2007). Finally, mechanical factors, such as chewing, have a con-
tradictory effect; the act of chewing may promote calculus formation
by increasing salivary flow rate (Dawes, 1970), whereas chewing abra-
sive materials may mechanically remove calculus deposits (Gaar, Rølla,
& van der Ouderaa, 1989).
Besides the multifactorial nature of calculus formation processes
and the challenge this can cause to the interpretation of the embedded
micro-debris, additional issues arise from the fact that the morphological
variation of teeth, expressed in the co-existence of smooth surfaces,
fissures and pits, creates different surface morphologies that facilitate
or hinder calculus formation (Hillson, 2005). Furthermore, within each
plaque deposit there may be marked variation in pH, nutrient availabil-
ity, and temperature, which can affect the formation and structure of
the calculus (Hillson, 2005).
2.2 | Relevant aspects for archeology
There are three main aspects of the dental calculus formation process
most relevant to archeology. First, and most importantly, the process
of calculus formation ceases at death, therefore it has high archaeologi-
cal integrity as postmortem inclusions of micro-debris from soil are
very unlikely (Middleton & Rovner, 1994).
The second aspect, and the one mostly considered to date, is that
dental calculus can entrap debris, microorganisms, molecules of various
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| 73
types that enter the mouth during the life of an individual, providing a
novel archaeological record (e.g., Buckley et al., 2014; Radini, Buckley,
et al., 2016; Radini, Nikita, et al., 2016; Warinner, Rodrigues, et al.,
2014; Warinner, Hendy, et al., 2014).
Thirdly, the rate of calculus formation is variable and associated
with differences in diet among individuals, salivary flow rate, health of
the individual, local pH and genetic factors (Marcotte & Lavoie, 1998).
There continues to be no clear understanding of when and how fast
dental calculus builds up on the teeth of different individuals, or how
much of an individual’s life may be represented in this matrix, which
means that comparative and quantitative approaches are currently not
possible (e.g., Leonard, Vashro, O’Connell, & Henry, 2015; Power,
Salazar-García, Straus, Morales, & Henry, 2015).
Despite this limitation, the direct connection of dental calculus to
individual ingestion as well as its ubiquity in past populations means
that it has extraordinary potential for investigating details of past lives,
both at individual and population levels.
3 | DENTAL CALCULUS AND ANCIENT DIET
In archeology, the study of dental calculus has been approached mac-
roscopically (quantity and location of calculus deposits on teeth), micro-
scopically (debris entrapped in it), and more recently, biomolecularly.
Early work on dental calculus focused on macroscopic quantification
and location of calculus found (Dobney & Brothwell, 1987) and this
approach is still sometimes used (e.g., Humphrey et al., 2014; Jankaus-
kas & Palabeckaite, 2006; Keenleyside, 2008; Lillie, 1996). Different
recording protocols have been proposed for this purpose, ranging from
simple presence/absence (e.g., Belcastro et al., 2007) to more detailed
ordinal schemes (Brothwell, 1981; Dobney & Brothwell, 1987) or even
continuous schemes that measure the maximum extent of the deposit
(Hillson, 2000). Such studies generally treat the amount of dental calcu-
lus per individual, combined with observations on other dental diseases
such as caries, as dietary indicators as well as markers of oral health
and oral hygiene. A major limitation in this approach is that dental cal-
culus deposits, particularly the supragingival ones, can detach from the
teeth both during life and postdepositionally (Buikstra & Ubelaker,
1994), which can bias the results. Detachment is less common for sub-
gingival deposits as these are protected to some extent by the gum
(Figure 3), unlike supragingival deposits, which accumulate on the
exposed surfaces of the teeth.
The potential of dental calculus to be a trap for a variety of micro-
scopic dietary and environmental micro-debris was first recognized in
1975 when phytoliths, opaline silica deposits that form in certain types
of plants, were extracted from samples of ungulate dental calculus
(Armitage, 1975). Dobney and Brothwell (1987, 1988) were the first to
explore the potential for obtaining information on archaeological sam-
ples of dental calculus, using scanning electron microscopy (SEM) to
identify pollen grains, phytoliths, charcoal, microscopic fragments of
cereal chaff and animal hairs from a range of different historical and
archaeological material. Following this, using SEM, fossilised bacteria
were detected in the calculus matrix of Neanderthal (Pap, Tillier,

74 | RADINI
Arensburg, Weiner, & Chech, 1995; Vandermeersch et al., 1994), Natu-
fian (Arensburg, 1996) and multi-period Chilean samples (Linossier,
Gajardo, & Olavarria, 1996). A more recent SEM analysis identified
microfossils related to diet, including palm phytoliths while diatoms
were used to examine water sources in an Easter Island population
(Dudgeon & Tromp, 2012)
Samples of dental calculus were first decalcified to extract the
embedded microfossils, which were then studied using light microscopy
from the extinct ape Gigantopithecus blacki, in order to identify the pres-
ence of embedded phytoliths (Ciochon, Piperno, & Thompson, 1990).
This was followed by Middleton & Rovner (1994), who identified phyto-
liths and starch granules from samples of herbivores. Subsequently,
starch granules (Juan-Tresserras, Lalueza, Albert, & Calvo, 1997; Scott
Cummings & Magennis, 1997) and phytoliths (Lalueza Fox, Juan, &
Albert, 1996) were extracted from human samples while a range of phy-
toliths was recovered from a sample of mastodon calculus (Gobetz &
Bozarth, 2001). Capasso, Di Tota, Jones, and Tuniz (1995) evaluated the
utility of synchrotron radiation microprobe analysis with potentially
promising results, though this method has yet to be developed further.
While many of these early publications highlighted the potential for
extracting useful paleodietary information from dental calculus, none
attempted to identify the specific sources of the microfossils.
Since 2008, the analysis of material remains entrapped in dental
calculus has increased significantly. While this has largely focused on
the extraction and identification of microfossils, in particular starch
granules and phytoliths which emerge from the dental calculus matrix
as it is decalcificed, a range of morphological and analytical methods
are now used to access the material entrapped in the calculus (Hardy
et al., 2012; Power, Salazar-García, Wittig, & Henry, 2014; Warinner,
Rodrigues, et al., 2014; Warinner, Hendy, et al., 2014). However, the
largest number of publications describes microfossil extraction and
identification using optical microscopy. Most studies have focused on
the identification of starch granules, and to a lesser extent phytoliths
(e.g., Henry, Brooks, & Piperno, 2014; Henry & Piperno, 2008; Henry
et al., 2012; Horrocks, Nieuwoudt, Kinaston, Buckley, & Bedford,
2013; Li et al., 2010; Mercader, 2009; Mickleburgh & Pag nez,
an-Jime
2012; Piperno & Dillehay, 2008; Tao et al., 2015; Wang, Fuller, Wei,
Chang, & Hu, 2015; Wesolowski, Ferraz Mendonça de Souza,
Reinhard, & Ceccantini, 2010). The principal aim of these studies has
been to identify dietary components, though food processing through
evaluation of starch granule alteration has also been explored (Henry,
Brooks, & Piperno, 2011; Henry, Hudson, & Piperno, 2009). However,
the complexity of starch granule alteration has been highlighted, and
suggests that further work is required to explore potential diagenetic
processes (Barton & Torrence, 2015; Collins & Copeland, 2011).
Since 2015, an increasing number of papers have highlighted
problems regarding the uncritical identification and interpretation of
the data, most notably in terms of the integrity of the link between
diet and the microfossils found, and their identification (Buck, Berb-
esque, Wood, & Stringer, 2015; Buck & Stringer, 2014; Leonard et al.,
2015; Power et al., 2015; Radini, Buckley, et al., 2016; Wang et al.,
2015). It has also become clear that accumulation of debris in calculus
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ET AL.
is more random than previously thought, which suggests that sampling
as a proxy for full reconstruction is problematic. In terms of starchy
food, it is clear that starch granules present in dental calculus do not
represent dietary breadth though they may be more reliable at the
population level (Leonard et al., 2015) while Power et al (2015) also
detected dietary correlations at the level of populations, using phyto-
liths, though not with the starch granules. This suggests that broad
interpretations of diet based solely on microfossil material should be
viewed with caution.
The morphological study of all archaeological plant-based materials
is based on comparison with reference collections. In relation to this,
the need for a better understanding of taphonomic processes affecting
starch granule survival is as applicable to material extracted from dental
calculus as it is to the analysis of other archaeological materials, such as
residues extracted from stone tools (Barton & Torrence, 2015). Like-
wise, although the preservation of starch granules is not in itself prob-
lematic due to the integrity of the mineralized matrix of calculus
through time, the overlapping size and shape of starch granules that
occur among and between species of plants, even in terms of widely
used domesticated plants such as millets (Lucarini, Radini, Barton, &
Barker, 2016; Wang et al., 2015), means use of starch granule morphol-
ogy alone for detailed species identification is also problematic.
A range of analytical techniques have been used, often in combina-
tion with microscopy, to further characterize entrapped material. A
study that combined scanning electron microscopy (SEM) with elemen-
tal analysis using energy-dispersive X-ray spectroscopy (EDX) has
shown potential in detecting food and micro-debris linked to occupa-
tional habits (e.g., Charlier et al., 2010); however, it does not recover
and identify as many particles as light microscopy (Power et al., 2014).
Inductively coupled plasma mass spectrometry (ICP-MS) analysis of
trace elements in dental calculus samples also seems promising and it
has so far been used in the identification of carbohydrates and fish
(Lazzati et al., 2016).
Stable isotope analysis has also been investigated for its potential
to study the composition of materials embedded in dental calculus as a
proxy for ancient dietary reconstruction (Scott & Poulson, 2012). How-
ever, Salazar-García, Richards, Nehlich, and Henry (2014) have demon-
strated that the isotopic values obtained from dental calculus are not
equal to those from bone collagen, possibly due to the diversity of
material entrapped in the calculus and the variable amounts of calculus
accumulation in individuals. This means the results obtained do not
reflect a life-time average, as is the case with bone collagen. However,
Wang et al. (2015) correlated carbon isotope data taken from samples
of bone collagen, with starch granules extracted from dental calculus.
Thermal desorption–gas chromatography–mass spectrometry (TD–
GC–MS) and pyrolysis–gas chromatography–mass spectrometry (Py–
GC–MS) can be used to characterise a wide range of organic materials
in dental calculus. The mineral component of dental calculus traps
organic compounds and provides them with a protective environment,
which allows biological marker compounds (biomarkers) characteristic
of the original source to survive. The identification of specific chemical
compounds enabled Hardy et al. (2012) to detect exposure of

RADINI ET AL.
n, Spain, to wood
Neanderthal individuals from the site of El Sidro
smoke, bitumen or oil shale and a range of plant items providing the
earliest direct evidence for the use of medicinal plants in human history.
Ingestion of the plant Cyperus rotundus L., was detected throughout the
multi period sequence at Al Khiday, Sudan (Buckley et al., 2014). Today,
this plant is considered primarily as a problematic weed in many coun-
tries (Sivapalan, 2013) suggesting that dental calculus has the potential
to recover evidence for foods and medicinal plants that have been for-
gotten. The earliest direct evidence for plant foods in the genus Homo
has been identified at the 1.2 million year old site of Sima del Elefante,
Atapuerca, Spain (Hardy et al., 2016) while chemical evidence for plant-
based nutrients was identified at the 300,000–400,000 years old Lower
Palaeolithic site of Qesem Cave, Israel (Hardy et al., 2015).
Dental calculus is also a source of bacterial genetic material and
holds potential as a biomolecular reservoir (Preus, Marvik, Selvig, & Ben-
nike, 2011). A number of papers have reviewed the recent application of
biomolecular techniques to the study of ancient dental calculus and its
relevance to ancient diet and oral and overall health (Huynh, Verneau,
Levasseur, Drancourt, & Aboudharam, 2016; Metcalf, Ursell, & Knight,
2014; Pacey, 2014; Weyrich, Dobney, & Cooper, 2015). De la Fuente,
Flores, and Moraga (2012) amplified DNA and identified bacteria from
4000-year-old samples from Chile. Similarly, Adler et al. (2013) identified
a wide range of oral bacteria, demonstrating a shift in their composition
as carbohydrates from domesticated plants became prominent. Warin-
ner, Rodrigues, et al. (2014) applied shotgun DNA sequencing to samples
of historic age dental calculus. This study highlighted the link between
oral pathogens, host immunity and dietary patterns by identifying in the
oral environment, opportunistic pathogens, antibiotic resistant genes,
bacterial and human proteins, as well as DNA sequences from dietary
sources. Only a portion of the material identified using biomolecular evi-
dence has been securely linked to diet (e.g., Warinner, Hendy, et al.,
2014). However, much of this evidence, such as consumption of leafy
crops of the Brassicaceae family traditionally have a low archaeological
visibility (Warinner, Rodrigues, et al., 2014; Warinner, Hendy, et al.,
2014). Warinner et al. (2015) also detected the first direct evidence of
milk consumption by identifying the protein b-lactoglobulin (BLG) in
human dental calculus from the Bronze Age (ca. 3000 BCE).
The use of biomolecular techniques may be limited in some cases,
such as for the earlier stages of the Palaeolithic, due to the apparent deg-
radation of biomolecular evidence in the deep past (e.g., Hardy et al.,
2016), but they appear to hold great potential for more recent samples.
4 | MICROPARTICLES IN DENTAL
CALCULUS AS A RESULT OF
NON-DELIBERATE CONSUMPTION
There is little doubt that much of the material extracted from dental
calculus represents items that found their way into the mouth during
life due to deliberate ingestion. However, the human mouth is a recipi-
ent of countless other particles and debris, and dental calculus has the
potential to trap any material that reaches the mouth by incorporating
and embedding it into its mineralised matrix.
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| 75
4.1 | Gastrophagy
Buck et al. (2015) suggest gastrophagy, or the eating of the stomach
contents of an animal, may confound palaeodietary reconstruction. In
this respect they suggest that chemical compounds indicative of yar-
 n Nean-
row and camomile found in the dental calculus of the El Sidro
derthals (Hardy et al., 2012) were the outcome of the eating of the
stomach contents of an animal that had previously eaten these plants
rather than the result of deliberate ingestion of non-nutritious plants
for self-medication (Buck et al., 2015). In fact, herbivores graze on a
wide variety of different plants and often actively avoid these strong
tasting plants (Hardy, Buckley, & Huffman, 2016). There is however,
extensive evidence for chyme consumption among recent and modern
hunter-gatherers who tend to make use of all the parts of killed animals
(Sinclair, 1953) and its use among prehistoric hunter-gatherers has
already been hypothesised (Speth, 2010). Chyme can provide valuable
nutrients, in terms of partially digested plant materials; for example
among the Inuit it provided access to lichens that had been made edi-
ble through partial digestion, and which formed an essential part of
their diet particularly in winter when there was a lack of other plant-
based resources (Sinclair, 1953). As phytoliths are silica bodies, and
therefore are reasonably resistant to degradation, it is possible that
they could endure through the partial digestion process that forms
chyme, and then become embedded in dental calculus (Henry et al.,
2014). For example, phytoliths have been found in samples of copro-
lites (e.g., Horrocks, Irwin, McGlone, Nichol, & Williams, 2003).
4.2 | Secondary eating of debris embedded
or settled on food or drinks
Dust and dirt, including grit particles settled on food, can reach the
mouth accidentally and become embedded in the dental calculus
matrix (Blatt et al., 2011; Buckley et al., 2014; Charlier et al., 2010;
Hardy et al., 2015, 2016). Soil particles can also be accidentally
ingested on food. A good example of this can be found in Dudgeon
and Tromp (2012) who retrieved large quantities of globular echinate
palm phytoliths (found in many parts of palm plants, including fruits,
fruit stones but also leaves) together with evidence for sweet potato
tubers from samples of dental calculus taken from Rapa Nui. However,
no palm species that produced such phytoliths were present in the
location at the time the studied individuals lived (Tromp & Dudgeon,
2015) and it later emerged that the palm phytoliths may have survived
in soil and became embedded in the skin of the edible tubers as they
grew (Tromp & Dudgeon, 2015). This study illustrates the potential
pitfalls of assuming that materials found in dental calculus are auto-
matically the result of deliberate ingestion of food items or diet.
4.3 | The human mouth as a “dust trap”:
pathways of inclusion by inhalation
In humans, as in all other land animals, air inhalation occurs constantly.
Breathing through the mouth occurs during speaking, eating, communi-
cating, panting after exertion and when the nose is blocked by nasal
mucus. It is safe to assume that many prehistoric and historic

76 | RADINI
populations were exposed to high levels of smoke and soot, thus
organic and inorganic “dust” and particles could have become embed-
ded in dental calculus. Dust, which can be caused naturally by wind or
generated by human activity, can produce high concentrations of
breathable debris. Se, Mei, Inthavong, and Tu (2010) demonstrated
that particles up to 70 lm are habitually inhaled through the mouth
while larger particles can also be inhaled under certain conditions. The
production of dust, often incorporating large amounts of particulate
matter below 100 lm, is characteristic of a great variety of traditional
human industries including stone-working, pottery manufacture, grain
storage, certain types of food production such as grinding and cooking
with flour and wood working. Such debris is considered a health hazard
in modern societies, since particles below 10 lm can reach the lungs
(Pope & Dockery, 2006). Respiratory health and air quality in past soci-
eties have received little or no attention so far, in part due to the pau-
city of evidence (Brimblecombe, 2011); however, exposure to smoke
and pollutants is likely to extend deep into the human past (Makra,
2014; Naeher et al., 2007; Hardy et al., 2015, 2016). Histological
assessment of the lungs of the Tyrolean Ice Man (Oetzi) which dates to
5400 cal. BP, as well as ancient human mummies (Egyptian, Peruvian,
and Aleutian), has shown that anthracosis (a pathology of the lungs
associated with prolonged exposure to smoky environments), has been
a regular disorder since ancient times (Mirsadraee, 2014; Zimmerman
et al., 1981). In this respect, a wide range of mineral and organic “dust”
from materials such as traditional pigments are also known (Murr,
2009).
Examples of environmental material found in dental calculus, and
most likely the result of inhalation, include insect parts, pollen, micro-
charcoal, soot (which most likely represents smoke inhalation), minute
grit and dust particles, plant fibers and phytoliths potentially derived
from cultural practices (Blatt et al., 2011; Buckley et al., 2014; Hardy
et al., 2015, 2016; Radini, Buckley, et al., 2016), or from accidental
ingestion, for example, from bedding during sleep. An example of acci-
dental inhalation/ingestion are the fungal debris such as spores and
hyphae, which have been found in several studies (e.g., Hardy et al.,
2015, 2016; Radini, Nikita, et al., 2016; Warinner, Hendy, et al.,
2014). Fungal spores detected in dental calculus samples have been
considered as food items (Afonso-Vargas, La Serna-Ramos, & Arnay-
de-la-Rosa, 2015; Power et al., 2015). Hardy et al. (2015) highlight
the ubiquity of fungal spores in the environment, and suggest that
they may well result from accidental ingestion or inhalation. The
fruiting bodies of fungal spores may become useful food items such
as mushrooms; however, assuming that fungal spores in calculus are
the result of deliberately ingested mushrooms needs to be viewed
with caution.
4.4 | The role of extramasticatory uses of teeth
A further source of non-dietary debris in the mouth is the use of teeth
in non-masticatory activities (e.g., Blatt et al., 2011). Physical evidence
for the non-masticatory use of teeth, including tooth wear is extremely
common in prehistoric, historic and some modern human populations
from across the world (Bonfiglioli, Mariotti, Facchini, Belcastro, & Con-
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ET AL.
demi, 2004; Eshed, Gopher, & Hershkovitz, 2006; Hinton, 1981; Loz-
ano, Mosquera, de Castro, Arsuaga, & Carbonell, 2009; Lukacs &
Pastor, 1988; Molnar, 2008; Ryan & Johanson, 1989; Volpato et al.,
2012). The use of teeth to hold, soften or shred material, as well as in
grooming activities and leaf chewing is widespread in the ethnographic
record (Hardy, 2008, 2016). One example comprises an Inuit popula-
tion with extreme dental wear among the anterior teeth, which was
linked to the preparation of walrus and seal hides for clothing, making
threads out of animal sinew (Clement & Hillson, 2012; Mayhall, 1976;
Pedersen, 1947). Non-dietary chewing of leaves, such as tobacco or
betel nut chewing in humans, or leaf-rolling in the mouth of chimpan-
zees (e.g., McLennan & Huffman, 2012) could explain the presence of
calcium oxalate in the dental calculus of primates found in recent stud-
ies (Power et al., 2014).
4.5 | Food preparation, food stickiness, oral clearance,
and oral hygiene
Food preparation practices can significantly alter the structure and
nutritious quality of plants (Butterworth, Ellis, & Wollstonecroft, 2016)
and can also have important effects on dental calculus in terms of the
formation and survival of micro-debris. For example, starch granules
and phytoliths can be altered and degraded by the use of grinding tools
in food preparation (e.g., Lucarini et al., 2016). The use of grinding tools
can incorporate grit and stone particles into the food which accelerate
tooth wear and can also lead to particles becoming embedded in dental
calculus. In addition chewing starchy food, even when uncooked, frees
the starch granules from their enclosing amyloplasts enhancing the
effects of amylase in the saliva, which results in the further breakdown
of these granules (BeMiller & Whistler, 2009).
The way food preparation practices may affect the formation of
calculus has been given little consideration. As already discussed,
chewing abrasive materials can mechanically remove calculus deposits
(Gaar et al., 1989) but the way food is prepared and its relative softness
affects dental calculus build up just as it affects the rate of dental wear.
Several studies have demonstrated the association between food proc-
essing and dental wear levels, whereby processed soft-textured food
with low fiber content results in less dental wear (Deter, 2009; Eshed
et al., 2006; Molnar, 1971). Alternatively, the ingestion of sand, grit and
other hard particles that become embedded in the food during grind-
ing, drying and other food processing practices, results in increased
dental wear (e.g., Lev-Tov Chattah & Smith, 2006; Smith, 1984). How-
ever, the correlation between dental calculus build-up and preservation
and dental wear as a proxy for food processing practices, has yet to be
explored.
In addition to its potential effect on dental calculus preservation,
cooking tends to alter the properties of various foodstuffs and other
inclusions, rendering many of them often impossible to identify in den-
tal calculus deposits. Proteins become denatured and various food
borne pathogens are killed (Carmody & Wrangham, 2009; Gaman &
Sherrington, 1996; Radley, 1968; Svihus, Uhlen, & Harstad, 2005; Tes-
ter, Qi, & Karkalas, 2006). The effect of cooking on starch granules is
well established, and results in their disruption and gelatinisation at

RADINI ET AL.
which point they are no longer visibly present as microfossils in dental
calculus, though a small amount of starch granules are resistant to deg-
radation and can survive (Radley, 1968). In some cases, particularly
when cooked using traditional open cooking methods, some food does
not “cook through.” In this case, not all starch granules disrupt and can
survive intact after having been cooked using outside ovens (Buckley
et al., 2014; Schnorr, Crittenden, & Henry, 2016; Thoms, Laurence,
Short, & Kamiya, 2015).
The roles of food stickiness, salivary clearance and oral hygiene are
also relevant when evaluating potential for diet on the basis of micro-
fossils entrapped in calculus, because such aspects may affect the
quantity of dental calculus build up as well as the typology of debris.
Food stickiness refers to the ability of certain foods to adhere to dental
and other oral surfaces (Lucas, Prinz, Agrawal, & Bruce, 2004). Many
factors determine stickiness, including water content in the food, its
viscosity, as well the nature of the food ingredients (e.g., sugars of low
molecular weight generate increased stickiness, whereas carbohydrates
and proteins of high molecular weight tend to minimize stickiness, see
review in Adhikari, Howes, Bhandari, & Truong, 2001). Food stickiness
is an important aspect to be taken into consideration during dental cal-
culus studies as particles of stickier food may well be more likely to
become embedded in dental calculus, though further work is required
to investigate the implications of this in terms of dietary reconstruc-
tions using dental calculus.
Clearance from the oral cavity is one of the most important sali-
vary functions since it removes food and dirt and maintains correct pH
and oral biofilm in the oral cavity (Humphrey & Williamson, 2001).
Different food types (sugars and fat) clear at different rates, though no
direct link between food stickiness and speed of clearance has been
detected (Dawes et al., 2015). The extent to which food-related micro-
debris can become incorporated into the calculus matrix is likely to
depend on how long and how much the food and other debris stick
to the tooth and how fast each category of ingested debris takes to
be swallowed and removed from the mouth. Therefore the nature of
food preparation may further affect the archaeological record
entrapped in the calculus matrix, though further work is needed to
address this.
The property of food stickiness, as well as fibrous debris that can
become entrapped between teeth, generate the need for oral hygiene
practices. Dental hygiene is neither a product of modern society, nor
an exclusively human phenomenon. Chimpanzees have been recorded
cleaning each-other’s teeth using their fingers and sticks (McGrew &
Tutin, 1973). Japanese macaques use their hair or the hair of another
individual to floss between their teeth (Leca, Gunst, & Huffman, 2010),
while long-tailed macaques in Thailand which interact intensively with
humans, have been observed to use human hair as flossing material
(Watanabe, Urasopon, & Malaivijitnond, 2007).
Among prehistoric human populations, a well-known case pro-
posed as evidence for oral hygiene activities are the interproximal
grooves on hominin teeth (Brothwell, 1963) for which tooth picking
has long been suggested as a cause (Ubelaker, Phenice, & Bass, 1969).
Interproximal grooving has been found on samples of all hominin
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| 77
species since Homo habilis (Puech & Gianfarani, 1988) and the grooves
are widespread among Neanderthal populations (Dąbrowski et al.,
2013; Estalrrich et al., 2011; Formicola, 1988; Frayer & Russell, 1987;
Lozano, de Castro, Carbonell, & Arsuaga, 2008; Urbanowski et al.,
2010; Villa & Giacobini,1995). Many materials have been suggested as
tooth picks including wood, and grass (Brown & Molnar, 1990; Eck-
hardt & Piermarini, 1988; Hlusko, 2003). While a number of hypothe-
ses have been put forward that attempt to explain interproximal
grooves, tooth picking to extract food stuck in between teeth is still
rez-
considered to be the most likely cause (Ungar, Grine, Teaford, & Pe
rez, 2001). Grass contains abundant phytoliths and it has been sug-
Pe
gested that with prolonged use, these are sufficiently abrasive to cause
the grooves (Hlusko, 2003).
The use of chewing sticks to maintain oral hygiene is widespread
in traditional societies (Almas, 2002; Idu, Umweni, Odaro, & Ojelede,
2009; Jose, Sharma, Shantaram, & Ahmed, 2011). Many of the plants
used for chewing have antibacterial qualities, which may aid oral
hygiene (Idu et al., 2009; Jose et al., 2011). In a Dakshina Kannada pop-
ulation from India for example, 25 different plants are used in oral
hygiene, either being chewed, or applied to the mouth. Each plant has
a specific role, including treatment of oral ulcers, gum disease, tooth
decay, toothache, caries and stomatitis. Other materials, including char-
coal, soot, clove oil, ghee, honey, and salt are also used to clean teeth
and relieve pain (Jose et al., 2011). All of these could potentially leave
chemical traces or microfossils embedded in dental calculus. Likewise,
oral hygiene practices are likely to have had an impact on the preserva-
tion of dental calculus, as they may remove or reshape deposits. The
possibility that plant parts and grass were used in oral hygiene suggests
that phytoliths found in dental calculus need to be viewed with an
open mind as to their origin, and that reference collections need to be
sufficiently broad to incorporate other potential sources, in addition
to food. Finally, oral hygiene may be responsible for the removal or
remodelling of dental plaque during life therefore reconstructions of
diet need to be conducted with great caution as the extent of a
person’s life that is reflected in the calculus deposit is unknown. Two
recent dental calculus studies have noted the presence of non-edible
wood fibers with interproximal grooves; in one case Radini, Buckley,
et al. (2016) reported chemical compounds and microdebris derived
from non-edible conifer wood fragments embedded in Neanderthal
calculus samples adjacent to teeth with extensive tooth wear and
 n, Spain, while
morphological evidence for tooth picking from El Sidro
Hardy et al. (2016) also detected non-edible wood fiber from a
sample of dental calculus from the 1.2 million year old Sima del
Elefante site.
4.6 | Archaeological evidence of non-dietary
remains in human dental calculus
A number of studies have demonstrated that non-dietary micro-debris
is preserved in the calculus matrix of archaeological samples. Blatt et al.
(2011) recovered evidence of cotton fibers (Gossypium sp.) in dental
calculus samples from Danbury, Ohio, dated 900–1100 AD. Charcoal
debris and mineral “grit” were found in the dental calculus of a Brazilian

78 | RADINI
Sambaqui population, though this was interpreted as possible contami-
nation (Wesolowski et al., 2010). Charlier et al. (2010) found, in addi-
tion to probable food debris, plant fibers possibly generated by the use
of the mouth as a third hand, as well as some mineral debris/crystals,
possibly from sand and grinding stones, which may have been either
inhaled or ingested. Elemental analysis of some of the crystals trapped
in dental calculus identified debris derived from the environment in
several individuals and possibly work-related pollution in one individual
from the Etruscan population of Monterenzio Vecchia (Charlier et al.,
2010). Hardy et al. (2012) identified evidence for smoke inhalation and
bitumen which may be related to tool hafting. Buckley et al. (2014)
identified microcharcoal and numerous plant fibers that suggest the
non-masticatory use of the teeth in a prehistoric Sudanese population
with extensive non-masticatory dental wear, while Hardy et al. (2015)
retrieved evidence of exposure to potential respiratory irritants in sam-
ples of Lower Palaeolithic hominins. Radini, Nikita, et al. (2016) also
found evidence of plant fibers consistent with flax and/or hemp in den-
tal calculus samples from Medieval Leicester (UK).
Consideration of all of these lines of evidence points to a degree
of complexity and multiple potential pathways by which material can
become entrapped in human dental calculus. It is not currently clear
whether the limited published information on non-dietary debris is the
result of their absence in the samples, or because this debris has not
been identified or reported.
5 | CONCLUSIONS
The research on ancient human dental calculus has significantly
increased, as the potential for reconstructing aspects of diet has
become apparent. However, while the potential of dental calculus to
entrap micro-remains of dietary origin has been recognized, the link
between such debris and the deliberate consumption of plants by
ancient people and their role in diet is more complex than previously
thought. Through a number of pathways, such as accidental ingestion,
oral breathing and the use of the teeth as a third hand, the human
mouth experiences a continued ingress of organic and inorganic par-
ticles that are present in the environment or derived from human activ-
ities. Though the many ways debris can become embedded in dental
calculus may limit its usefulness in the detailed reconstruction of
ancient diet, they increase its value for obtaining wider biographical
information on past individuals. The approach of combining microscopy
with various analytical techniques and osteoarchaeological parameters
can help to differentiate between dietary and non-dietary debris (Rad-
ini, Buckley, et al., 2016). Likewise, given the presence of material that
is not related to diet, reference collections, which form the basis of all
identification of material remains, should incorporate non-dietary
plants and plant parts, including those used in material culture and oral
hygiene, as well as atmospheric and environmental contaminants.
One of the problems in understanding the sources of the material
embedded in dental calculus is the difficulty in conducting experimental
or modern comparative work. The use of live traditional populations in
this respect can potentially be problematic as it requires invasive
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ET AL.
research techniques and may encourage or support calculus build up,
which is detrimental to health. Therefore ethics must be taken in
account when working with live human populations, though experi-
mental archeology may help to clarify the flux of particles in the mouth.
Likewise, more research on recent archaeological populations for which
there is greater information available, will help to enhance our under-
standing of the extent to which dental calculus is representative of the
daily life of the studied individuals (Radini, Nikita, et al., 2016).
The study of dental calculus has come a long way since its potential
for informing on past human and hominin lives was first recognised. The
quality and value of information that can be obtained from the extraction
and identification of embedded microfossil material, and the recovery
and identification of all microfossil material, covers a much wider range
of information than simply diet. Recovery of all material requires very
gentle decalcification methods while identification of the material can be
challenging. Likewise, to maximise this information and identify material
that is unequivocally linked to diet, the material remains also need to be
contextualised within a broader framework. Conducted in this way, the
analysis of microfossils extracted from dental calculus and the recogni-
tion of their potential to provide environmental and cultural, as well as
dietary information will ensure this relatively new subdiscipline develops
to provide a wide range of exciting data and information on past lives
and environments, within realistic parameters.
ACKNOWLEDGMENTS
We would like to thank two anonymous reviewers and the associate
editor for their valuable comments. Efthymia Nikita’s contribution to
this paper was supported by a Marie Skłodowska-Curie Individual
Fellowship [Programme/Call: H2020—H2020-MSCA-IF-2015, Pro-
posal: 702991—HumAn].
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